ATR is a multifunctional regulator of male mouse meiosis

Widger, Alexander; Mahadevaiah, Shantha K.; Lange, Julian; ElInati, Elias; Zohren, Jasmin; Hirota, Takayuki; Pacheco, Sarai; Maldonado-Linares, Andros; Stanzione, Marcello; Ojarikre, Obah A.; Maciulyte, Valdone; Rooij, Dirk G. de; Tóth, Attila; Roig, Ignasi; Keeney, Scott; Turner, James M. A.

doi:10.1038/s41467-018-04850-0

Cited by 52 publications

(75 citation statements)

References 79 publications

(107 reference statements)

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“…ATM negatively regulates DSB induction by SPO11 7 . Despite the fact that Atm -/spermatocytes harbor a 10-fold increase in DSBs 7 , RAD51/DMC1 foci form at similar levels as in WT while RPA foci counts are increased in mutant cells 38 . One possible reason why the number of foci do not correlate with the large increase in DSBs could be that recombinase levels or activity is limiting for filament formation.…”

Section: Rad51 Dmc1 and Atm Negatively Regulate Long-range Resectiomentioning

confidence: 88%

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Paiano

Yamada

et al. 2019

Preprint

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Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11. The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations ("hotspots") are determined by the DNA sequence specificity of PRDM9. Here, we demonstrate the first direct detection of meiotic DSBs and resection in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum lengths of resected DNA, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate ("SPO11-RI") present at all hotspots. The SPO11-bound intermediate is dependent on PRDM9 and caps the 3' resected end during engagement with the homologous template. We propose that SPO11-RI is generated because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm -/spermatocytes, SPO11-RI is reduced while unresected DNA-bound SPO11 accumulate because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing. MainRecombination between homologous chromosomes during meiosis requires DNA double-strand break (DSB) formation by the topoisomerase-like protein SPO11 1 . After cutting, SPO11 remains covalently bound to a two-nucleotide, 5' overhang at both ends of the DNA via phosphotyrosyl linkage. Recombination then begins with the processing of SPO11-bound DSBs into resected 3' single-stranded DNA (ssDNA) tails that preferentially invade the homologous chromosome by the recombinases DMC1 and RAD51. Studies in budding yeast Saccharomyces cerevisiae determined that the MRE11/RAD50/NBS1 (MRN) complex detects SPO11 and cooperates with Sae2 to produce a nick on the SPO11-bound strand via MRE11 endonuclease activity 2 . The nick serves as an entry point for both short-range MRE11 3'-5' exonuclease activity to degrade back to the DSB, thereby removing covalently bound SPO11 attached to a ssDNA oligonucleotide, as well as for more extensive long-range processing of 5' strands (Extended Data Fig. 1a) 2 . In budding yeast, Exo1 nuclease is uniquely responsible for this long-range 5'-3' resection 3 .Moreover, short-and long-range resection are tightly coupled in a single processive reaction (Extended Data Fig. 1a). As a result, meiotic DSBs are maximally resected as soon as they appear and unresected SPO11-bound DSBs are extremely rare 4-6 . While ATM has been shown to regulate DSB numbers and locations 7,8 , its remains unclear whether it also functions downstream in regulating SPO11 processing and resection.Distinct from yeast, DSB hotspots in mice and humans are determined by the DNA binding specificity of the PRDM9 methyltransferase 9 . Besides positioning DSBs, PRDM9...

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Section: Rad51 Dmc1 and Atm Negatively Regulate Long-range Resectiomentioning

confidence: 88%

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Paiano

Yamada

et al. 2019

Preprint

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“…In mammals, ATR localises to meiotic chromosomes [20] and is a key regulator of meiotic events. ATR deletion in male mice causes fragmentation of the chromosome axis, and ATR is required for synapsis and loading of recombinases RAD51 and DMC1 at DSB sites [21,22]. In Drosophila , loss of Mei-41, the fly ATR orthologue, not only abrogates checkpoint signalling [23], but also alters the frequency and spatial distribution of COs [24].…”

Section: Introductionmentioning

confidence: 99%

Separable roles of the DNA damage response kinase Mec1(ATR) and its activator Rad24(RAD17) within the regulation of meiotic recombination

Crawford

Cooper

Marsolier‐Kergoat

et al. 2018

Preprint

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13 During meiosis, programmed DNA double-strand breaks (DSBs) are formed by the 14 topoisomerase-like enzyme, Spo11, activating the DNA damage response (DDR) kinase 15 Mec1 ATR via the checkpoint clamp loader, Rad24 RAD17 . At single loci, loss of Mec1 and Rad24 16 activity alters Spo11-dependent DSB formation and recombination outcome, but their genome-17 42 the generalised pathway, DNA damage leads to the activation of Tel1 ATM (Ataxia Telengiectasia-43 Mutated) and Mec1 ATR (Rad3-related) checkpoint kinases (Human orthologues are indicated 44with superscript text), via the Mre11-Rad50-Xrs2 NBS1 -complex and Rad24 RAD17 (reviewed in [2]). 45Rad24 RAD17 is the loader of the Ddc1 RAD9 -Rad17 RAD1 -Mec3 HUS1 ("9-1-1") checkpoint clamp, that 46 binds to the ssDNA/dsDNA junctions that arise following resection of DNA ends [3]. Tel1 ATM and 47 Mec1 ATR modulate downstream targets via Rad53 CHK2 [4], causing cell cycle arrest and the 48 modulation of transcription [5]. Notably, in mammalian cells, RAD17 and ATR have both 49 overlapping and distinct functions in the DDR. RAD17 is required for the recruitment of RAD9 50 after DNA damage, a function that it performs even in the absence of ATR [6]. Conversely, ATR 51 can localize to sites of DNA damage independently of RAD17, and can also be activated by 52 other factors such as RPA-coated ssDNA [7], suggesting separable and complementary roles 53 for ATR and RAD17.54 55 In meiosis, Spo11-DSBs initiate homologous recombination and are therefore essential for the 56 generation of crossovers (COs) between homologous chromosomes. In most organisms, 57 including mammals and S. cerevisiae, the organism utilised in the work presented here, such 58 recombination-dependent interactions facilitate homologue pairing during leptotene-zygotene, 59 full alignment and connection via the synaptonemal complex at pachytene, and subsequent 60 reductional chromosome segregation at the meiosis I nuclear division [8]. In S. cerevisiae 61 meiosis, similar to the vegetative DDR pathway, the 9-1-1 clamp complex, its loader Rad24, and 62 the Mre11-Rad50-Xrs2 complex act as damage sensors [9], with ssDNA, produced by the 63 resection of Spo11-DSBs, activating Mec1 [10]. By sensing ongoing recombination activity and 64 unrepaired DSBs, Mec1 acts as a molecular rheostat to modulate the progression of meiotic 65 prophase via the Ndt80 transcription factor, and the Mek1 kinase, a paralogue of Rad53 (CHK2 66 in mammals; [10-13]). Due to this transient checkpoint activation, Mec1 is able to prolong the 67 stage during which Spo11-DSB formation can occur [14], and both Mec1 and Rad24 promote 68 CO formation and suppress ectopic (non-allelic) recombination [15,16]. The interaction between 69 Mec1 and the 9-1-1 complex also contributes to other Mec1 functions such as phosphorylation 70 of the meiosis-specific chromosome axis protein, Hop1 (HORMAD1/2 in mammals [17]), which 71is important for the maintenance of homologue bias and chiasma formation [18,19]. In mammals, 72 Crawford et al. 2018 3 ATR local...

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“…As mentioned earlier, female Seckel mice have been proposed to have meiotic recombination defects (Murga et al, 2009), since we have previously demonstrated that ATR function is required to complete meiotic recombination in spermatocytes Widger et al, 2018), we studied meiotic prophase progression in Seckel mouse oocytes.…”

Section: Meiotic Prophase Progression Is Not Altered In Seckel Mouse mentioning

confidence: 98%

ATR function is indispensable to allow proper mammalian follicle development

Pacheco

Garcia-Caldés

Roig

2018

Preprint

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Mammalian female fertility relies on the proper development of follicles. Right after birth in mouse, oocytes associate with somatic ovarian cells to form follicles. These follicles grow during adult lifetime to produce viable gametes. In this study, we analyzed the role of the ATM and rad3-related (ATR) kinase in mouse oogenesis and folliculogenesis using a hypomorphic mutation of the Atr gene (Murga et al., 2009). Female mice homozygote for this allele have been reported to be sterile. Our data show that female meiotic prophase is not grossly altered when ATR levels are reduced. However, follicle development is majorly compromised since Atr mutant ovaries present a decrease of growing follicles. Comprehensive analysis of follicular cell death and proliferation suggest that wild-type levels of ATR are required to achieve optimal follicular development. Altogether, these findings suggest that reduced ATR expression causes sterility due to defects in follicular progression rather than in meiotic recombination. We discuss the implication of these findings for the use of ATR inhibitors as anti-cancer drugs and its possible side effects on female fertility.

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ATR is a multifunctional regulator of male mouse meiosis

Cited by 52 publications

References 79 publications

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis

Separable roles of the DNA damage response kinase Mec1(ATR) and its activator Rad24(RAD17) within the regulation of meiotic recombination

ATR function is indispensable to allow proper mammalian follicle development

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